Poly-gamma-glutamic acid as a novel material for

viscosupplementation and treating OA of the knee – A pilot

study

Authors: Albert Prescott, Dr. Louis R. Stock, and Dr. Victor Shen

Disclosure:

Study funded by Crescent Innovations Inc.

Presented by Al Prescott, President and Founder

Helping Manage PainHelping Manage Pain

OsteoarthritisOsteoarthritis

IncurableIncurable and and chronicchronic form of arthritis. form of arthritis. Characterized by a breakdown of cartilage Characterized by a breakdown of cartilage

and synovial fluid in the joints.and synovial fluid in the joints. Resulting in (or caused by) inflammation.Resulting in (or caused by) inflammation. In U.S., 12% of US adults between the ages of In U.S., 12% of US adults between the ages of

25 and 74 years. 25 and 74 years. Globally, prevalence estimates were 1-80%, Globally, prevalence estimates were 1-80%,

although most were 5-25%.although most were 5-25%.

J Am Osteopath Assoc. 2001 Apr;101(4 Suppl Pt 2):S2-5.Semin Arthritis Rheum. 2014 Jun;43(6):713-20. doi: 10.1016/j.semarthrit.2013.12.007. Epub 2013 Dec 31.

Osteoarthritis – Risk FactorsOsteoarthritis – Risk Factors

AgeAge - 80% of persons older than 75 years - 80% of persons older than 75 years ObesityObesity - women who lost average of 11 - women who lost average of 11

pounds decreased their risk up to 50%pounds decreased their risk up to 50% Joint InjuryJoint Injury - Jobs requiring simultaneous - Jobs requiring simultaneous

squatting and turning may cause up to 30% squatting and turning may cause up to 30% of knee OA in men.of knee OA in men.

Muscle weaknessMuscle weakness Ethnic characteristicsEthnic characteristics Nutritional factorsNutritional factors GeneticsGenetics

J Am Osteopath Assoc. 2001 Apr;101(4 Suppl Pt 2):S2-5.Semin Arthritis Rheum. 2014 Jun;43(6):713-20. doi: 10.1016/j.semarthrit.2013.12.007. Epub 2013 Dec 31.Weight loss reduces the risk for symptomatic knee osteoarthritis in women. Ann Intern Med 1992;166:535-539

Osteoarthritis – Nonpharmacologic TreatmentsOsteoarthritis – Nonpharmacologic Treatments

Weight lossWeight loss Physical therapyPhysical therapy Occupational therapyOccupational therapy Patient educationPatient education Aerobic conditioning exercisesAerobic conditioning exercises Assistive devices/footwearAssistive devices/footwear

J Am Osteopath Assoc. 2001 Apr;101(4 Suppl Pt 2):S2-5.

Osteoarthritis – Pharmacological TreatmentsOsteoarthritis – Pharmacological Treatments

Systemic treatment modalitiesSystemic treatment modalitiesNonopioid analgesicsNonopioid analgesicsNonsteroidal anti-inflammatory drugsNonsteroidal anti-inflammatory drugsOpioid analgesicsOpioid analgesicsGlucosamine and chondroitin sulfateGlucosamine and chondroitin sulfate

Inra-articular therapyInra-articular therapyCorticosteroidsCorticosteroidsViscosupplementationViscosupplementation

Topical analgesicsTopical analgesics Joint ReplacementJoint Replacement

ViscosupplementationViscosupplementation

All have 1 key ingrediant – Hyaluronic Acid / Sodium Hyaluronate

Adverse Reactions – Joint Pain/swelling

Product/Study Hyaluronic Acid Placebo

Hyalgan 23% (1.8x) 13%

Synvisc One 4.9% (3.3x) 1.5%

Orthovisc 2.5% (1.3x) 2.0%

Why? All hyaluronic acids are either:

Avian tissue extracted, orFermented via bacterial pathogen.

Viscosupplementation - PerformanceViscosupplementation - Performance

All Made with Hyaluronic Acid, and all perform...All Made with Hyaluronic Acid, and all perform...

...the same.the same.Rheumatology 2002;41:1240–1248

Viscosupplementation Viscosupplementation

In the event that an alternate material could be In the event that an alternate material could be shown to be: shown to be: Non-inflamatory, Non-inflamatory, Non-immunogenic, Non-immunogenic, Non-cytotoxic, ANDNon-cytotoxic, ANDAble to reduce inflammationAble to reduce inflammation

Chronic OsteoarthritisChronic Osteoarthritis

InflammationInflammation

Inflammation can be measured by collagenase MMPs (1,8, 13*)

New MaterialNew Material

How do we propose to reduce How do we propose to reduce inflammation?inflammation?

gPGA – poly-gamma-glutamic acid

gPGA

gPGA was produced by fermenting a non-pathogenic gPGA was produced by fermenting a non-pathogenic Bacillus. Bacillus.

gPGA was purified via filtration and buffer exchange gPGA was purified via filtration and buffer exchange and formulated using sterile saline for injection at a and formulated using sterile saline for injection at a concentration of 30 mg/ml. concentration of 30 mg/ml.

The molecular weight was determined to be 1.22 The molecular weight was determined to be 1.22 million Daltons with a polydispersity of 1.1 using million Daltons with a polydispersity of 1.1 using HPLC and MALLS. HPLC and MALLS.

gPGA viscosity was measured at 4,900 cps on a gPGA viscosity was measured at 4,900 cps on a Brookfield viscometer using a CP-52 spindle at an Brookfield viscometer using a CP-52 spindle at an rpm of 0.5rpm of 0.5

Pre-Clinical Safety DataPre-Clinical Safety Data

Have Passed ISO Standars for:Have Passed ISO Standars for:CytotoxicityCytotoxicitySensitization / IrritationSensitization / IrritationIntracutaneous – DirectIntracutaneous – DirectSystemic ToxicitySystemic ToxicityRabbit PyrogenRabbit PyrogenGenotoxicityGenotoxicityHemocompatabilityHemocompatabilityHemolysis Hemolysis ImplantationImplantationGenerally Regarded as Safe - Generally Regarded as Safe - GRASGRAS

MMP Study Design

18 rats were divided into 3 groups of six. 18 rats were divided into 3 groups of six. All rats had their left knee injected with monoiodoacetate All rats had their left knee injected with monoiodoacetate

(MIA)[Kim 2012]. (MIA)[Kim 2012]. 7 days later the rats were treated with a single 50 uL 7 days later the rats were treated with a single 50 uL

injection, in that knee, of either saline, Hyalgan®(a injection, in that knee, of either saline, Hyalgan®(a commercially approved HA), or gPGA. commercially approved HA), or gPGA.

At Day 9, all animals were sacrificed, the hind limbs At Day 9, all animals were sacrificed, the hind limbs disarticulated, and the cartilage removed.disarticulated, and the cartilage removed.

Collagenase MMPs were extracted using a two step Collagenase MMPs were extracted using a two step procedure described in Janusz. procedure described in Janusz.

Results were reported as equivalent MMP-1 activity per Results were reported as equivalent MMP-1 activity per mg cartilage.mg cartilage.

ResultsResults

* *

Note: Hyaluronic Acid group contained a single animal that tested at 17.8 MMP1/mg cartilage

2.96 2.65 2.06, P<0.05

Additional Thoughts - Outlier

It is possible that the single large MMP value in the Hyalgan It is possible that the single large MMP value in the Hyalgan group is evidence of an adverse reaction to Hyalgan. group is evidence of an adverse reaction to Hyalgan.

It is known that HA is associated with elevated adverse It is known that HA is associated with elevated adverse reaction rates in the human population compared to saline reaction rates in the human population compared to saline treatments. treatments.

Hyalgan had 23% of the patients (38 out of 164) reported pain at the injection site, as Hyalgan had 23% of the patients (38 out of 164) reported pain at the injection site, as compared to 13% (22 out of 168) in the saline placebo treated group. compared to 13% (22 out of 168) in the saline placebo treated group.

23% out of 6 animals is 1.38, so on the surface, having 1 animal with elevated levels of an 23% out of 6 animals is 1.38, so on the surface, having 1 animal with elevated levels of an OA related MMP might not be out of the question. OA related MMP might not be out of the question.

Remains speculation since there is no corroborating evidence of adverse reactions of HA in Remains speculation since there is no corroborating evidence of adverse reactions of HA in rats. rats.

If, however, we accept that elevated MMPs are a marker of inflammation, and the single If, however, we accept that elevated MMPs are a marker of inflammation, and the single value that is almost 10x the average of the group constitutes an adverse reaction, then it is value that is almost 10x the average of the group constitutes an adverse reaction, then it is worth noting that the PGA group does NOT contain a similar point. This may suggest that worth noting that the PGA group does NOT contain a similar point. This may suggest that PGA has a better safety profile that HA for treating OA of the knee.PGA has a better safety profile that HA for treating OA of the knee.

More work is needed.More work is needed.

Additional Thoughts – Contralateral Data

Additional Thoughts – Chicken or Egg?

Why does viscosupplementation work?Why does viscosupplementation work?– Mechanical?Mechanical?

– Biological?Biological?

– Other?Other?

Efficacy – Traditional Thoughts

Justification for Viscosupplementation Justification for Viscosupplementation

(mechanical)(mechanical)CushioningCushioningLubricationLubricationReplacement of degraded hyaluronic acidReplacement of degraded hyaluronic acid““The results indicate that proteoglycan subunit has a The results indicate that proteoglycan subunit has a clearance half life of around 12 hours in normal joints and clearance half life of around 12 hours in normal joints and that this rate is not significantly altered in the presence of that this rate is not significantly altered in the presence of an active synovitis.” Page-Thomas.an active synovitis.” Page-Thomas.““Pulse chase experiments of intracellular hyaluronan Pulse chase experiments of intracellular hyaluronan suggest the half-life of internalized HA is 2-3 hours.” Tammisuggest the half-life of internalized HA is 2-3 hours.” Tammi

Efficacy – Crescent Innovations' Thoughts

Degenerative Joint DiseaseDegenerative Joint DiseaseInjuryInjuryInflammationInflammationJoint DegradationJoint Degradation

Efficacy – Crescent Innovations' Thoughts

Inflammatory MarkersInflammatory Markers– CytokinesCytokines

– Matrix-metallo-protinasesMatrix-metallo-protinases

All ProteinsAll Proteins

Efficacy – Crescent Innovations' Thoughts

Hyaluronic Acid, and Poly-g-glutamic acidHyaluronic Acid, and Poly-g-glutamic acid

Efficacy – Crescent Innovations' Thoughts

Efficacy – Crescent Innovations' Thoughts

gPGAgPGA– Neg. charge per glutamic acid (MW=128)Neg. charge per glutamic acid (MW=128)

HA HA – Neg. Charge per disaccharide (MW=360)Neg. Charge per disaccharide (MW=360)

gPGA HAS TRIPLE CHARGE DENSITYgPGA HAS TRIPLE CHARGE DENSITY

StatusStatus

Completed “Behavioral Modification Study”Completed “Behavioral Modification Study” Preparing for Clinical TrialsPreparing for Clinical Trials

QuestionsQuestions

Al Prescott, PresidentAl Prescott, President

978-764-8604978-764-8604

alprescott@crescentinnovations.comalprescott@crescentinnovations.com